化学发光法和酶联免疫法作为筛选试验测定丙肝抗体的评价

目的比较化学发光法(CLIA)和酶联免疫法(EIA)测抗-HCV对诊断HCV感染的检出率,分析CLIA测定抗-HCV作为HCV感染初筛试验的重要临床意义。方法分别用CLIA、EIA方法检测所有临床标本中的抗-HCV,选取抗-HCV初筛阳性样本进行HCV RNA确认试验。结果 6461例样本中,EIA和CLIA测抗-HCV阳性率分别为2.86%和3.17%;确认试验中,CLIA法抗-HCV阳性与HCV RNA的符合率为97.1%,显著高于EIA组(91.9%)。EIA法测抗-HCV,S:CO>5.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为69.7%;CLIA法测抗-HCV,S:CO>2.0以上,与HCV RNA检测符合率可达100%;弱阳性样本,与HCV RNA符合率为92.9%。有31例样本CLIA法抗-HCV和HCV RNA阳性,而EIA法抗-HCV阴性;有4例样本EIA法检测为阳性,而CLIA法和PCR确认试验均为阴性。结论CLIA法测抗-HCV作为HCV感染初筛实验,与传统EIA相比,特异性和阳性预测值更高,有效降低了假阳性率,有利于临床医生对HCV感染患者的早期诊断和治疗。     

流式荧光免疫技术检测抗核抗体谱的性能与临床应用评价

目的 分析流式荧光免疫技术(FFIA)检测自身抗体谱的结果及评价其在系统性红斑狼疮(SLE)诊断中的应用价值。方法 用流式荧光免疫法和免疫印迹法同时检测219例自身免疫病患者(包括92例SLE患者和127例非SLE的其他自身免疫病患者)、231例其他疾病患者、50例健康体检者血清的特异性自身抗体(抗nRNP、Sm、SS-A、SS-B、Jo-1、Scl-70、rRNP、PCNA、Nucleosome、Histone、CenP-B、M2、dsDNA),计算两种方法的阴阳符合率,并采用Kappa检验评估结果一致性;采用受试者工作特征曲线(ROC)比较各抗体在系统性红斑狼疮诊断中的应用价值。结果 FFIA与免疫印迹法检测各项自身抗体总符合率在87%至98%之间,Kappa值在0.179至0.854之间,其中抗CenP-B抗体一致性最好(Kappa值=0.854),抗His抗体一致性最差(Kappa值=0.179)。在219例自身免疫病例组数据中,根据试剂盒给定的阳性结果判断标准得到的免疫印记法灵敏度(62.0%)高于流式荧光(44.6%),而特异性略低于后者,分别为89.8%和94.4%,但二...     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

化学发光酶免疫分析法在乙肝病毒及核心抗体定性检测中的应用分析

目的 通过对比,探讨了化学发光酶免疫分析法在乙肝病毒及核心抗体定性检测中的应用价值.方法 选取2013年5月至2015年5月在我院的疑似乙型肝炎患者80例,抽取空腹血液样本后都分别进行乙肝病毒以及核心抗体的化学发光酶免疫分析法及ELISA法检测,并对其检测结果进行分析.结果 化学发光酶免疫分析法检出乙型肝炎病毒阳性78例,检出率为97.5％;而ELISA检出乙型肝炎病毒阳性76例,检出率为95.0％,两种方法的检出率对比差异无统计学意义(P＞0.05).化学发光酶免疫分析法对于乙肝病毒核心抗体IgM与IgG的检测阳性率分别为80.0％和70.0％,而ELISA法检测两种抗体的阳性率则分别为18.8％和20.0％,化学发光酶免疫分析法对乙肝核心抗体IgM与IgG的检测阳性率明显高于ELISA法(P＜0.05).ELISA法检出HBc-IgM的最低限为0.135 IU/ml,检出HBc-IgM最低限为0.143 IU/ml;化学发光酶免疫分析法检出HBc-IgM最低限为0.032 IU/ml,检出HBc-IgG最低限为0.038 IU/ml.结论 化学发光酶免疫分析法在乙型肝炎检测中具有高的检出率,尤其对乙肝病毒的核心抗体的检测敏感度较高,值得在临床推广应用.     

两种试剂检测SARS冠状病毒IgG抗体的结果分析

目的用两种试剂盒检测本院医务人员和住院患儿血清中SARS冠状病毒(SARS-CoV)IgG抗体以比较两种盒的检测结果.方法采用北京华大和本院研制的两种酶联免疫吸附法(ELISA)试剂盒,对923例医务人员和150例住院患儿血清进行SARS-CoV IgG抗体检测.结果 923例医务人员中,北京华大盒检测阳性10例,阴性913例;本院研制盒检测阳性20例,阴性903例,两组差异无显著性(p>0.05).两种试剂盒检测150例住院患儿血清全部阴性.结论两种试剂盒检测结果无显著差异.150例住院患儿血清中无SARS冠状病毒IgG抗体.     

ELISA检测百日咳抗体的初步研究

以干菌为抗原作ELISA,检测血清百日咳IgG抗体,最适包被菌液浓度为20亿/ml。以血清稀释度≥1:320,OD_(490nm)≥0.47(阴性对照血清OD+3S)为阳性判定标准,测得抗体滴度和阳性检出率均高于凝集法。非百日咳病人血清用ELISA检测交叉率为5.0％;对细菌凝集滴度<1:80的正常人血清的假阳性率为2.0％。以阻断试验证实本法特异性强,稳定性好,且试验所需血清量仅为5μl。     

两种检测方法测定抗甲状腺球蛋白抗体和抗甲状腺过氧化物酶抗体结果的比较

目的 应用化学发光免疫分析法（CLIA）和放射免疫分析法（RIA）分别测定血清抗甲状腺球蛋白抗体（抗TgAb）和抗甲状腺过氧化物酶抗体（抗TPOAb）.方法 采用CLIA法和RIA法测定304例不同类型甲状腺疾病患者和38名健康对照者血清抗TgAb和抗TPOAb水平.结果 CLIA法测定血清抗TgAb和抗TPOAb水平与RIA法测定的结果具有良好的一致性.结论 CLIA法测定血清抗TgAb和抗TPOAb可以用来替代RIA法.     

两种试剂检测SARS冠状病毒IgG抗体的结果分析

目的　用两种试剂盒检测本院医务人员和住院患儿血清中SARS冠状病毒 (SARS -CoV)IgG抗体以比较两种盒的检测结果 .方法　采用北京华大和本院研制的两种酶联免疫吸附法 (ELISA)试剂盒 ,对 92 3例医务人员和 15 0例住院患儿血清进行SARS -CoVIgG抗体检测 .结果　 92 3例医务人员中 ,北京华大盒检测阳性 10例 ,阴性 913例 ;本院研制盒检测阳性 2 0例 ,阴性 90 3例 ,两组差异无显著性 (p>0 .0 5 ) .两种试剂盒检测 15 0例住院患儿血清全部阴性 .结论　两种试剂盒检测结果无显著差异 . 15 0例住院患儿血清中无SARS冠状病毒IgG抗体 .     

不同化学发光分析系统检测丙型肝炎病毒抗体的性能评价

目的 评价罗氏Cobas e601及雅培Architect i2000两种化学发光免疫分析系统检测抗-HCV的分析性能和一致性.方法 按照CLSI推荐的方法对两台分析仪检测抗-HCV的精密度、检出限、阴阳性符合率等性能进行验证试验;两台仪器分别检测120例有反应性血清样本及30例无反应性样本,用Kappa检验评价检测结果的一致性.结果 两台仪器的精密度良好;检出限均为0.3NCU;阴阳性符合率为100％;两个分析系统检测抗-HCV总体符合率为70.67％,无反应性、e601 S/CO≥15.0、i2000 S/CO≥6.0样本结果的符合率为100％,e601 S/CO为1.01 ～5.0和i2000 S/CO为1.01 ～2.0样本结果的符合率为0和44.4％:经统计分析κ=0.35在Kappa系数区段中一致性程度属于弱(κ=0.35,P＜0.05).结论 两个分析系统检测抗-HCV的分析性能均良好,适用于临床标本的常规检测,对于两台仪器检测结果不一致的样本,尤其是e601 S/CO 1 ～ 15及i2000 S/CO 1～6的样本,建议进行补充实验(RIBA,NAT)来明确诊断.     

IgG型抗E抗体联合IgG型抗Fyb抗体的血清学检测分析

目的 分析IgG型抗E抗体联合IgG型抗Fyb抗体的血清学检测结果及配血对策.方法 对患者血标本进行ABO及Rh血型鉴定和不规则抗体筛选,对抗体筛选阳性血标本采用盐水法、间接抗人球蛋白法进行抗体特异性鉴定.确定抗体特异性后,再用抗人球蛋白法检测红细胞是否含有相应抗原;采用盐水法、凝聚胺法及间接抗人球蛋白法进行交叉配血试...     

High-quality, cost-effective strategy for detection of autoantibodies to extractable nuclear antigens

We evaluated methods for the detection of autoantibodies to extractable nuclear antigens (ENAs) to determine the strategy that yielded the most cost effective and clinically meaningful result. We prospectively compared counterimmunoelectrophoresis (CIEP) with and without serum prediffusion (SPD) and found that SPD significantly improved the quality of precipitation lines. This resulted in a decreased requirement for repeat testing and, consequently, was associated with a significant decrease in reagent costs and specimen turnaround time. We also retrospectively compared reactivity by CIEP, CIEP plus SPD, enzyme-linked immunosorbent assay (ELISA), and line immunoassay (LIA) of 52 serum samples that were previously determined to be positive for ENAs, and we correlated the results with clinical diagnoses. There was significant agreement among CIEP, CIEP plus SPD, ELISA, and LIA for the detection of anti-SS-A, anti-SS-B and anti-RNP. In general, CIEP, CIEP plus SPD, and LIA correlated better with the clinical diagnoses than ELISA, even though ELISA detected anti-ENAs more often than the other methods. CIEP plus SPD is therefore the most cost effective method for the identification of clinically meaningful ENAs. Based on our experience, we now screen for ENAs by CIEP, and positive samples are then typed by CIEP plus SPD. Samples that are difficult to interpret are then further assessed by an alternative method.     

High-Quality, Cost-Effective Strategy for Detection of Autoantibodies to Extractable Nuclear Antigens

We evaluated methods for the detection of autoantibodies to extractable nuclear antigens (ENAs) to determine the strategy that yielded the most cost effective and clinically meaningful result. We prospectively compared counterimmunoelectrophoresis (CIEP) with and without serum prediffusion (SPD) and found that SPD significantly improved the quality of precipitation lines. This resulted in a decreased requirement for repeat testing and, consequently, was associated with a significant decrease in reagent costs and specimen turnaround time. We also retrospectively compared reactivity by CIEP, CIEP plus SPD, enzyme-linked immunosorbent assay (ELISA), and line immunoassay (LIA) of 52 serum samples that were previously determined to be positive for ENAs, and we correlated the results with clinical diagnoses. There was significant agreement among CIEP, CIEP plus SPD, ELISA, and LIA for the detection of anti-SS-A, anti-SS-B and anti-RNP. In general, CIEP, CIEP plus SPD, and LIA correlated better with the clinical diagnoses than ELISA, even though ELISA detected anti-ENAs more often than the other methods. CIEP plus SPD is therefore the most cost effective method for the identification of clinically meaningful ENAs. Based on our experience, we now screen for ENAs by CIEP, and positive samples are then typed by CIEP plus SPD. Samples that are difficult to interpret are then further assessed by an alternative method.     

Detection of anti-SS-A/Ro and anti-SS-B/La antibodies of IgA and IgG isotypes in saliva and sera of patients with Sj?gren's syndrome.

To assess the frequency and the possibility of local production of autoantibodies against SS-A/Ro and SS-B/La in patients with primary Sj?gren's syndrome (SS), serum and saliva samples were obtained from 42 patients with SS, 10 with rheumatoid arthritis without sicca syndrome, and 12 healthy volunteers. Autoantibodies were detected using enzyme-linked immunosorbent assay and immunoblotting. The frequencies of IgA anti-SS-A antibody, IgA anti-SS-B antibody, IgG anti-SS-A antibody and IgG anti-SS-B antibody in serum from SS patients were 45%, 50%, 43% and 21%, respectively. The frequencies of IgA anti-SS-A antibody, IgA anti-SS-B antibody, IgG anti-SS-A antibody and IgG anti-SS-B antibody in saliva from SS patients were 31%, 33%, 40%, and 19%, respectively. We also found secretory IgA anti-SS-A and anti-SS-B antibodies accompanying secretory components in saliva and sera in representative SS patients. Significant correlations were found between serum and salivary levels of IgA anti-SS-A antibodies, and between serum and salivary levels of IgA anti-SS-B antibodies in SS patients. Significant correlations were also found between serum and salivary levels of IgG anti-SS-A antibodies, and between serum and salivary levels of IgG anti-SS-B antibodies in SS patients. Immunoblot analysis confirmed the presence of IgA-class autoantibodies against SS-A and SS-B in saliva and serum from representative patients. The presence of IgA- and IgG-class autoantibodies against SS-A and SS-B and those accompanying secretory components in saliva from SS patients suggests the local production of these antibodies and the relationship between local and systemic antibody responses.     

Clinical and serological characteristics of systemic lupus erythematosus: 128 cases

OBJECTIVE: To analyse the clinical and serological characteristics of systemic lupus erythematosus (SLE) in the center of Tunisia. METHODS: We studied 128 patients with SLE aged one to 73 years. Antinuclear antibodies (ANA) were detected by an immunofluorescence method. Anti-double-stranded DNA (anti-dsDNA) antibodies, anti-extractable nuclear antigen antibodies (anti-Sm, anti-SS-A, anti-SS-B and anti-RNP) and anti-cardiolipin (aCL of IgG, IgA and IgM isotypes) antibodies were detected by ELISA. RESULTS: Malar rash (71%) and anemia (71%) were the most common clinical manifestations. Arthritis was seen in 62.5%. Severe kidney damage was observed in 39%. Pericarditis and pleuritis were observed in only 23%. Neurological manifestations (16%) were uncommon. Clinical manifestations of anti-phospholipid syndrome (SAPL) were observed in 15%. ANA were detected in 100%, anti-dsDNA in 76%, anti-Sm in 55.5%, anti-SS-A in 64%, anti-SS-B in 33.6%, anti-RNP in 49%. aCL of IgG, IgA and IgM isotypes were detected in 63.5%, 49% and 40.6% of the patients respectively. The only significant positive clinical associations were those of arthritis with anti-dsDNA antibodies (p = 0.022) and malar rash with anti-SS-A antibodies (p = 0.002). CONCLUSIONS: This study suggests that tunisians with SLE present, in general, a mild form of disease predominantly manifested by cutaneous, musculoskeletal and hematologic involvement but low prevalence of major organ damage.     

化学发光免疫法检测梅毒特异性抗体结果分析

目的 探讨化学发光法检测梅毒螺旋体特异性抗体的应用价值.方法 应用化学发光免疫测定法检测12503例住院患者梅毒螺旋体特异性抗体,并同ELISA、TPPA、RPR方法进行比较.结果 12503例住院患者化学发光法检出梅毒螺旋体特异性抗体阳性308例,同ELISA方法比较,阳性符合率99.08％,阴性符合率99.98％,总符合率99.93％.二者检测结果高度一致.同TPPA比较,阳性符合率98.7％.结论 化学发光免疫法检测梅毒特异性抗体具有特异性好、灵敏度高、结果易判断、便于自动化等优点,适合于梅毒的筛查.     

Prevalence in myositis of antibodies recognizing anti-U3 RNA probably in a novel complex with 22/25 kD protein and not fibrillarin.

New antibodies against a U3 snRNP, which were named anti-Myo 22/25 antibodies, were detected in four (8%) of 53 serum samples from patients with polymyositis/dermatomyositis (PM/DM) by RNA immunoprecipitation. In the protein immunoprecipitation analysis, all four serum samples precipitated 22 kDa and 25 kDa proteins, which were not precipitated by normal serum or serum positive for antifibrillarin antibodies. Three of the four PM/DM patients had other identified autoantibodies including anti-PL-12 antibodies, antihistone antibodies (AHA), anti-SS-A antibodies and anti-SS-B antibodies defined by double immunodiffusion, ELISA or RNA immunoprecipitation, although there were no significant correlations between anti-Myo 22/25 antibodies and clinical or laboratory findings. There may be a subgroup of PM/DM patients whose sera are positive for anti-Myo 22/25 antibodies.     

Comparison of counter immunoelectrophoresis with immunoblotting for detection of anti-extractable nuclear antigen antibodies in systemic lupus erythematosus

Anti-extractable nuclear antigen (ENA) antibodies were assayed by counter immunoelectrophoresis (CIE) and immunoblotting in patients with systemic lupus erythematosus (SLE). We found the two methods showed good concordance rates, the lowest being 67% for anti-SS-A. Immunoblotting was more sensitive in detecting anti-Sm, anti-SS-B and anti-PCNA (proliferating cell nuclear antigen); CIE was more sensitive for anti-nRNP and anti-SS-A. Overall, the prevalence of these anti-ENA antibodies in SLE was increased by 9-20% if immunoblotting was used in addition to CIE. Sera specific for the 52 kDa peptide of the SS-A antigen (anti-52kDa SS-A) were better detected by immunoblotting. Anti-PCNA antibody was found in 6.3% of SLE patients and was associated with active disease and hemolytic anemia. The positive rate of anti-Sm was 9% by CIE and 23.7% by immunoblotting and this antibody was a specific marker for SLE using either method. It was concluded that using immunoblotting in addition to CIE, the overall sensitivity of detection of anti-ENA antibodies in SLE was increased and clinically useful antibodies such as anti-52kDa SS-A and anti-PCNA could be detected.     

SS-B/La antigen purification, and ELISA detection of anti-SS-B/La antibodies in sera from patients with inflammatory connective tissue diseases

SS-B/La antigen was purified by immunoadsorbent columns with immunoglobulin from a patient with primary Sjögren's syndrome. Monitoring of the purification was facilitated by the fused rocket-immunoelectrophoresis technique. Technical ELISA variables for the detection of serum antibodies against the SS-B/La antigen were evaluated, and a recommended procedure is described. Prospective investigation of anti-SS-B/La antibodies in 103 blood donors and 131 patients with chronic inflammatory connective tissue diseases, including 43 patients with primary Sjögren's syndrome was performed. Anti-SS-B/La antibody concentrations were above normal in 65% of the patients with primary Sjögren's syndrome (verified by the strictly objective Copenhagen criteria for keratoconjunctivitis sicca and xerostomia) and 9% of patients with other chronic connective tissue diseases. The predictive value for primary Sjögren's syndrome among patients with increased levels of the anti-SS-B/La antibodies attending a rheumatology clinic was 78%.     

Differences in capabilities of different enzyme immunoassays to detect anti-hepatitis E virus immunoglobulin G in pigs infected experimentally with hepatitis E virus genotype 3 or 4 and in pigs with unknown exposure

Hepatitis E virus (HEV), a major cause of acute viral hepatitis in humans in many developing countries, is highly prevalent in the pig population worldwide. The objective of this study was to assess the capability of three porcine prototypes of a human enzyme-linked immunosorbent assay (ELISA), an in-house ELISA and a line-immunoassay (LIA) to detect anti-HEV antibodies in pigs infected experimentally with HEV (n = 57), known to be negative for HEV infection (n = 27), or with unknown exposure to HEV infection (field samples, n = 90). All 27 samples from non-infected pigs were negative with all five assays. The earliest detection of anti-HEV antibodies occurred at 14 days post-inoculation (dpi) with four of five assays. From 42 dpi, all samples from infected pigs were detected correctly as anti-HEV positive. Kappa analysis demonstrated substantial agreement among tests (0.62-1.00) at 14 dpi and complete agreement (1.00) at 56 dpi. The overall area under the curve for all quantitative tests as determined by receiver operator characteristic analysis ranged from 0.794 to 0.831 indicating moderate accuracy. The results showed that all five assays can detect anti-HEV IgG antibodies accurately in pigs infected experimentally with HEV. In field samples, a higher prevalence of anti-HEV IgG was found in breeding herds than in growing pigs (100% versus 66.7-93.9%). These serological assays should be very useful in veterinary diagnostic labs for HEV diagnosis in swine.